The present invention relates to image-enhancing agents, contrast agents or spectral shift agents to enhance tissue or organ images or nuclear spectra obtained from live animals with radioisotope scanning or NMR imaging or spectroscopy.
The imaging of internal structures and organs of live animals has been an important aspect of medicine since the advent of X-ray usage for this purpose. Among the techniques more recently developed for such imaging are those involving scanning for emission of particles from an internally located radioisotope. Such radioisotopes preferably emit gamma particles and are generally isotopes of metallic elements. One problem common to the diagnostic usage of such gamma particle-emitting radioisotopes concerns the localization of these materials at sites of particular interest rather than to have them randomly dispersed or rapidly excreted, by the kidney, for example. Another problem of such radioisotope mediated imaging concerns optimizing the circulating half-life of radioisotopes.
NMR intensity and relaxation images have been shown in recent years to provide another method of imaging internal structures and organs of live animals. Clinical magnetic resonance Imaging (MRI) is a rapidly growing, new form of brain and body imaging. Low-field (proton) MRI detects chemical parameters in the immediate environment around the protons of body tissues (predominantly water protons because of their relative abundance). Changes in these parameters occur very early in disease and are independent of physical densities detected by ionizing radiation. In the brain and central nervous system, MRI has allowed detection of tumors at an earlier clinical stage and with fewer imaging artifacts than is possible with computerized axial tomography (CAT) (Runge et al., (1983) Am. J. Radiol V 141, p 1209). Under optimal conditions, image resolution is in the submillimeter size range.
Six factors make it important to develop nontoxic MRI image-enhancing agents analogous to those available for CAT. 1. They increase the specificity of MRI diagnosis. 2. Smaller lesions can be identified earlier. 3. Image-enhancing agents enhance tumor masses differently than surrounding edema fluid or abscesses. This allows the extent and invasion of tumors to be defined more precisely. Lesions with infiltrative-type growth (e.g., certain metastatic carcinomas and glioblastomas) will require contrast agents for demarcation between tumor and edema fluid (Felix et al. (1985) Proc. Soc. Mag. Res. Med. V 2, p 831). 4. Image-enhancing agents improve the distinction between recurrent tumor and fibrous tissue resulting from surgery and radiation. 5. Image-enhancing agents can decrease the time required per scan and potentially decrease the number of scans required per procedure. This increases the volume of procedures and decreases their expense; and 6. Body imaging has a significantly lower resolution (typically 0.5-1.0 cm) and sensitivity (decreased signal-to-noise ratio) than brain imaging (Wesbey et al (1983) Radiology V 149, p 175). These differences result from the greater inhomogeneity of the magnetic field; the larger radiofrequency coil; unequal phase-pulsing of deep versus shallow nuclei; and motion artefacts produced by respiration, cardiac systole, gastrointestinal peristalsis, and voluntary muscle movement.
The discrete intensities of a two-dimensional, Fourrier-transformed image are described by the following general equation (for spin-echo pulse sequences): EQU Intensity=N(H).multidot.f (v).multidot.exp(-TE/T2).multidot.(1-exp(TE-TR/T1), where:
N(H)=number of protons in the discrete tissue volume (spin density); PA1 f(v)=a function of proton velocity and the fraction of protons which are moving (e.g., due to following blood); PA1 TE=time between the radio frequency (rf) pulse and the detection of signal (spin-echo); PA1 TR=the interval between repetition of the rf pulse. PA1 T1=the time interval associated with the rate of proton energy transfer to the surrounding chemical environment (spin-lattice relaxation); PA1 T2=the time interval associated with the rate of proton energy transfer, one to other (spin-spin relaxation).
T1 and T2 times have reciprocal effects on image intensity. Intensity is increased by either shortening the T1 or lengthening the T2. Tissue contrast occurs naturally and is related to variations in the chemical environments around water protons (major contributor) and lipid protons (usually minor). Chemical agents have been used to enhance this natural contrast. The one most widely tested clinically is the paramagnetic metal ion, gadolinium (Gd.sup.+3) (Runge et al. (1983) Am. J. Radiol V 141, p 1209 and Weinman et al. (1984) Am. J. Radiol V 142, p 619). Although gadolinium shortens both the T1 and T2 times, at the low dose used for clinical imaging, the T1 effect generally predominates and the image becomes brighter. Also, the rf pulse sequence can be programmed to accentuate T1 changes and diminish those due to T2 (Runge et al. (1983) Am. J. Radiol V 141, p 1209). Hence, "T1-weighted" enhancement can be achieved by selecting the most favorable Gd dose and pulse sequence.
The shortening of proton relaxation times by Gd is mediated by dipole-dipole interactions between its unpaired electrons and adjacent water protons. The effectiveness of Gd's magnetic dipole drops off very rapidly as a function of its distance from these protons (as the sixth power of the radius) (Brown (1985) Mag. Res. Imag. V 3, p 3). Consequently, the only protons which are relaxed efficiently are those able to enter Gd's first or second coordination spheres during the interval between the rf pulse and signal detection. This ranges from 10.sup.5 to 10.sup.6 protons/second ((Brown (1985) Mag. Res. Imag. V 3, p 3). Still, because Gd has the largest number of unpaired electrons (seven) in its 4f orbital, it has the largest paramagnetic dipole (7.9 Bohr magnetons) and exhibits the greatest paramagnetic relaxivity of any element (Runge et al. (1983) Am. J. Radiol V 141, p 1209 and Weinman et al. (1984) Am. J. Radiol V 142, p 619). Hence, Gd has the highest potential of any element for enhancing images. However, the free form of Gd is quite toxic. This results in part, from precipitation at body pH (as the hydroxide). In order to increase solubility and decrease toxicity, Gd has been chemically chelated by small organic molecules. To date, the chelator most satisfactory from the standpoints of general utility, activity, and toxicity is diethylenetriamine pentaacetic acid (DTPA) (Runge et al. (1983) Am. J. Radiol V 141, p 1209 and Weinman et al. (1984) Am. J. Radiol V 142, p 619). The first formulation of this chelate to undergo extensive clinical testing was developed by Schering-Berlex AG according to a patent application filed in West Germany by Gries, Rosenberg and Weinmann (DE-OS 3129906 A 1 (1981). It consists of Gd-DTPA which is pH-neutralized and stabilized with the organic base, N-methyl-D-glucamine (meglumine). The Schering-Berlex agent is nearing completion of Phase II clinical testing at selected centers across the United States and abroad. The results of preliminary studies indicate that almost all human brain tumors undergo significant enhancement (Felix et al. (1985) Proc. Soc. Mag. Res. Med. V 2, p 831 and K. Maravilla, personal communication). These include metastatic carcinomas, meningiomas, gliomas, adenomas and neuromas. Renal tumors are also enhanced satisfactorily (Lanaido et al. (1985) Proc. Soc. Mag. Res. Med. V2, p 877 and Brasch et al. (1983) Am. J. Radiol. V 141, p 1019). The Schering-Berlex formulation is projected to be available for general clinical use by late 1985.
Despite its satisfactory relaxivity and toxicity, this formulation has four major disadvantages.
(1) Chelation of Gd markedly decreases its relaxivity (by 1/2 an order of magnitude). This happens because chelators occupy almost all of Gd's inner coordination sites which coincide with the strongest portion of the paramagnetic dipole (Koenig (1985) Proc. Soc. Mag. Res. Med. V 2, p 833 and Geraldes et al. (1985) Proc. Soc. Mag. Res. Med. V 2, p 860).
(2) Gd-DTPA dimeglumine, like all small chelates, suffers a marked decrease in relaxivity at the higher proton Larmor frequencies used for clinical imaging (typically 15 Mhz) (Geraldes et al. 1985) Proc. Soc. Mag. Res. Med. V 2, p 860).
(3) Due to its low molecular weight, Gd-DTPA dimeglumine is cleared very rapidly from the bloodstream (1/2 in 20 minutes) (Weinman et al. (1984) Am. J. Radiol V 142, p 619). This limits the imaging window (to ca. 30 minutes); limits the number of optimal images after each injection (to ca. 2); and increases the agent's relative toxicity.
(4) A disproportionate quantity (&gt;90%) of Gd-DTPA is cleared by the kidneys Weinman et al. (1984) Am. J. Radiol V 142, p 619). Of much greater interest to MRI, are the abdominal sites involved in the early detection and staging of tumors (particularly the liver, and also the spleen, bone marrow, colon and pancreas).
Three approaches have been taken in attempts to overcome these disadvantages.
(1) Alternative, small chelating molecules have been tested. These make Gd more accessible to water protons but still chelate the metal with a sufficient affinity to potentially control its toxicity in vivo. The most effective of these chelators is DOTA, the polyazamacrocyclic ligand, 1,4,7,10-tetraazacyclododecane-N,N',N"-tetraacetic acid (Geraldes et al. (1985) Proc. Soc. Mag. Res. Med. V 2, p 860). Its relaxivity is approximately 2 times greater than that of Gd-DTPA over a wide range of Larmor frequencies. However, it is still less active than free Gd.
(2) Gd and Gd-chelates have been chemically conjugated to macromolecules, primarily the proteins, albumin (Brelman et al. (1981) Health Physics V 40, p 228 and Lauffer et al. (1985) Mag. Res. Imaging V 3, p 11), asialofetuin (Brelman et al. (1981) Health Physics V 40, p 228), and immunoglobulins (Lauffer et al. (1985) Mag. Res. Imaging V 3, p 11 and Brady et al. (1983) Soc. Mag. Res., 2nd Ann. Mtg., Works in Progress, San Francisco, Calif.). This increases the relaxivity of Gd by slowing its rate of molecular tumbling (rotational correlation time) (Lauffer et al. (1985) Mag. Res. Imaging V 3, p 11). This improves coupling of the energy-transfer process between protons and Gd (Geraldes et al. (1985) Proc. Soc. Mag. Res. Med. V 2, p 860, Lauffer et al. (1985) Mag. Res. Imaging V 3, p 11 and Brown et al. (1977) Biochemistry V 16, p 3883). Relaxivities are increased by multiples of 5 to 10 relative to Gd-DTPA (when compared as 1/T1 values at 1 millimolar concentrations of Gd) and by multiples of 2.5 to 5.0 (when compared as the molarities of Gd required to produce a specified decrease in the T1 relative to a control solution (physiologic saline).
The reasons for using the latter method of comparison are that 1) millimolar concentrations of Gd are never achieved in vivo--actual tissue concentrations achieved in the usual image enhancement are between 5 and 30 micromolar Gd; 2) the second method allows agents to be compared according to the more customary means of chemical activity radio, in other words, as the concentration required to produce a specified percentage decrease in the T1 (or T2) relaxation time. This latter method is the one used throughout the remainder of the application. The large drawback of conjugating DTPA to protein carriers for use in NMR image enhancement is that it has been difficult to stably conjugate more than 5 DTPA's (and hence Gd's) to each albumin molecule (Brelman et al. (1981) Health Physics V 40, p 228, Lauffer et al. (1985) Mag. Res. Imaging V 3, p 11 and Hnatowich et al. (1982) Int. J. Appl. Radiat. Isot. V 33, p 327 (1982).
Comparably low substitution ratios (normalized for molecular weight) have been reported for immunoglobulins (Lauffer et al. (1985) Mag. Res. Imaging V 3, p 11 and Brady et al. (1983) Soc. Mag. Res., 2nd Arn. Mtg., Works in Progress, San Francisco, Calif.) and fibrinogen (Layne et al. (1982) J. Nucl. Med. V 23, p 627). This results from the relative difficulty of forming amide bonds, the comparatively low number of exposed amino groups on typical proteins which are available for coupling, and the very rapid hydrolysis of DTPA anhydride coupling substrate which occurs in the aqueous solvents required to minimize protein denaturation during conjugation (Hnatowich et al. (1982) Int. J. Appl. Radiat. Isot. V 33, p 327 (1982) and Krejcarek et al. (1977) Biochem. Biophys. Res. Comm. V 77, p 581). The overall effect of these suboptimal conditions is that a large dose of carrier material is required to achieve significant in vivo effects on MR images. Indeed, low substitution ratios have generally limited the use of such protein-chelator-metal complexes to the more sensitive, radiopharmaceutical applications (Layne et al. (1982) J. Nucl. Med. V 23, p 627).
(3) Gd-DTPA has been entrapped in liposomes (Buonocore et al. (1985) Proc. Soc. Mag. Res. Med. V 2, p 838) in order to selectively enhance images of the reticuloendothelial organs (liver, spleen and bone marrow) and potentially the lungs. Liver clearance is mediated by phagocytic (Kupffer) cells which spontaneously remove these small (0.05 to 3 um) particles from the bloodstream (Buonocore et al. (1985) Proc. Soc. Mag. Res. Med. V 2, p 838). (Particles larger than 3 to 5 um are selectively localized in the lungs due to embolic entrapment in lung capillaries.) A recent report indicates that the small-sized Gd-liposomes produce effective decreases in liver T1's (as determined spectroscopically without imaging) (Buonocore et al. (1985) Proc. Soc. Mag. Res. Med. V 2, p 838). Also, insoluble Gd-DTPA colloids have recently been reported to enhance MR images of rabbit livers under in vivo conditions (Wolf et al. (1984) Radiographics V4, p 66). However, three major problems appear to limit the diagnostic utility of these devices. The multilamellar, lipid envelopes of liposomes appear to impede the free diffusion of water protons into the central, hydrophobic cores of these carriers, as assessed by the higher doses of Gd required for in vitro relaxivities equivalent to Gd-DTPA dimeglumine (Buonocore et al. (1985) Proc. Soc. Mag. Res. Med. V 2, p 838). This increases the relative toxicity of each Gd atom.
Even more importantly, these same lipid components cause the carriers to interact with cell membranes of the target organs. This leads to a marked prolongation of tissue retention (with clearance times of up to several weeks) (Graybill et al. (1982) J. Infect. Dis. V 145, p. 748 and Taylor et al. (1982) Am. Rev. Resp. Dis. V 125, p 610); and G. Kabala, personal communication). Two adverse consequences result. First, image enhancement does not return to baseline in a timely fashion. This precludes re-imaging at the short intervals (ca. 1-week) needed to assess disease progression and treatment effects. Second, significant quantities of the liposomally entrapped Gd-DTPA may be transferred directly into the membranes of host cells (Blank et al. (1980) Health Physics V 39, p 913; Chan et al. (1985) Proc. Soc. Mag. Res. Med. V 2, p 846). This can markedly increase the cellular retention and toxicity of such liposomal agents. The consequences for Gd toxicity have not yet been reported. Protein (albumin) microspheres with entrapped Gd and Gd chelates have been prepared and determined by the present applicant and others (Saini et al. (1985) Proc. Soc. Mag. Res. Med. V 2, p 896) to have only modest effects on T1 relaxivity in vitro. This is because most of the Gd as well as other entrapment materials (Widder et al. (1980) Cancer Res. V 40, p 3512) are initially sequestered in the interior of these spheres and are released very slowly as the spheres become hydrated (with t1/2's of hours) (Widder et al. (1980) Cancer Res. V 40, p 3512). This phenomenon has been found by the present applicant to markedly reduce the acute (30-to-90-minute) relaxivity of each Gd atom to approximately 1/10th that of Gd-DTPA dimeglumine. Hence, both the quantity of carrier material and the toxicity of Gd are both unnecessarily high.
Emulsions of insoluble, gadolinium oxide particles have been injected into experimental animals with significant image enhancing effects on the liver (Burnett et al. (1985) Magnetic Res. Imaging V 3, p 65). However, these particles are considerably more toxic than any of the preceding materials and are inappropriate for human use. Because of the significant disadvantages of existing MR image contrast agents, the present applicant has formulated improved, second-generation prototype agents with reduced toxicity, increased selectivity of organ uptake, as well as a significant potential for enhancing blood flow images.
Many of the advantages shown for the present developments concerning NMR image-enhancing agents (also referred to herein as NMR contrast agents or MR (magnetic resonance) contrast agents) are also expandable to other areas. For example, high-field NMR surface-coil spectroscopy of .sup.1 H .sup.13 C, .sup.19 F, .sup.23 Na, and .sup.31 P nuclei in spacially localized tissue volumes is gaining in importance and is starting to be applied experimentally to the noninvasive clinical monitoring of genetic and metabolic disorders; myocardial infarcts and metabolism; brain, liver and tumor metabolism; drug distribution and metabolism; blood flow and tissue perfusion measurements; and temperature monitoring in regional hyperthermia. Gadolinium and related agents can produce characteristic changes in the NMR spectrum of adjacent NMR-susceptible nuclei. These changes include: modulation of peak positions, widths, intensities, and relaxation rates (which affect intensity). Hence, perturbation of spectra by such chemical shift-relaxation agents can be used to localize and identify the source of NMR signals with respect to organ location, tissue compartment (intravascular versus extravascular), cell type within the tissue, and potentially, the specific metabolic pathways within cells which are altered by drugs and disease. Also in certain situations body scanning of radioisotopic emissions is particularly useful in achieving insight into internal structures. Most frequently the emissions scanned are those of metallic radioisotopes emitting gamma particles. The mode of administering these radioisotopic metals may have significant consequences on the internal localization and body half-life of these radioisotopes, leading to increased diagnostic usage of these emission scannings.